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Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

Ryan KA, Pimplikar SW - J. Cell Biol. (2005)

Bottom Line: APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm.In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined.Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

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AICD transgenic mice show barely detectable levels of AICD in the membrane fraction. (A) Cytosolic proteins from control (con) or two transgenic mice from indicated lines were separated on a 4–12% Bis-Tris NuPAGE gel, and the blots were probed with antibody 0443 after antigen retrieval as described in “Methods and materials.” Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). AICD band is clearly visible in FeCγ.25 mice (top panel, lanes 6 and 7), whereas a longer exposure shows the AICD protein present in FeCγ.12 mice (bottom panel, lanes 4 and 5), but not in control (lanes 2 and 3) or Fe.27 mice (lanes 8 and 9). (B) Brain homogenates from two control (con) transgenic mice from indicated lines were separated into cytosolic (Cyto.), membrane (Memb.), or nuclear (Nucl.) fractions, and the blots were probed with antibody 0443 as indicated above. Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). Although AICD was detected in the cytosol of only FeCγ transgenic mice (arrow), the membrane and nuclear fractions of all animals showed the AICD co-migrating band (arrows). (C) The AICD co-migrating band is absent in APP-KO mice. Brain membranes from two FeCγ.12 mice (lanes 2 and 3), APP-KO mice (lanes 4 and 5), and R1.40 transgenic mice expressing human APP with “Swedish mutation” (lanes 6 and 7) were probed with antibody 0443, as described above, on a NuPAGE gel. Top panel shows that the APP band is present in FeCγ.12 and in higher amounts in R1.40 mice, but is absent in APP-KO mice. Similarly, the middle panel shows the absence of APP-CTFs in the APP-KO mice. Note that the AICD co-migrating band (arrow) also is absent in APP-KO mice. (D) The AICD peptide becomes detectable only after antigen retrieval treatment (ART). Equal amounts of brain membrane proteins from two R1.40 mice were separated in duplicate on a NuPAGE gel. After electrophoretic transfer, the membrane was cut lengthwise to give two identical gels. One was exposed to boiling PBS for 5 min (ART), whereas the other was kept in PBS at room temperature (con). Arrow shows the AICD band, which is detectable only in the ART samples. (E) AICD band (arrow) is present in increased amounts in R1.40 transgenic mice compared with the controls (con) and is absent in APP-KO mice. Membrane fractions from indicated mice were probed with antibody 0443.
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fig2: AICD transgenic mice show barely detectable levels of AICD in the membrane fraction. (A) Cytosolic proteins from control (con) or two transgenic mice from indicated lines were separated on a 4–12% Bis-Tris NuPAGE gel, and the blots were probed with antibody 0443 after antigen retrieval as described in “Methods and materials.” Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). AICD band is clearly visible in FeCγ.25 mice (top panel, lanes 6 and 7), whereas a longer exposure shows the AICD protein present in FeCγ.12 mice (bottom panel, lanes 4 and 5), but not in control (lanes 2 and 3) or Fe.27 mice (lanes 8 and 9). (B) Brain homogenates from two control (con) transgenic mice from indicated lines were separated into cytosolic (Cyto.), membrane (Memb.), or nuclear (Nucl.) fractions, and the blots were probed with antibody 0443 as indicated above. Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). Although AICD was detected in the cytosol of only FeCγ transgenic mice (arrow), the membrane and nuclear fractions of all animals showed the AICD co-migrating band (arrows). (C) The AICD co-migrating band is absent in APP-KO mice. Brain membranes from two FeCγ.12 mice (lanes 2 and 3), APP-KO mice (lanes 4 and 5), and R1.40 transgenic mice expressing human APP with “Swedish mutation” (lanes 6 and 7) were probed with antibody 0443, as described above, on a NuPAGE gel. Top panel shows that the APP band is present in FeCγ.12 and in higher amounts in R1.40 mice, but is absent in APP-KO mice. Similarly, the middle panel shows the absence of APP-CTFs in the APP-KO mice. Note that the AICD co-migrating band (arrow) also is absent in APP-KO mice. (D) The AICD peptide becomes detectable only after antigen retrieval treatment (ART). Equal amounts of brain membrane proteins from two R1.40 mice were separated in duplicate on a NuPAGE gel. After electrophoretic transfer, the membrane was cut lengthwise to give two identical gels. One was exposed to boiling PBS for 5 min (ART), whereas the other was kept in PBS at room temperature (con). Arrow shows the AICD band, which is detectable only in the ART samples. (E) AICD band (arrow) is present in increased amounts in R1.40 transgenic mice compared with the controls (con) and is absent in APP-KO mice. Membrane fractions from indicated mice were probed with antibody 0443.

Mentions: We next determined the AICD levels by Western blot following a protocol (see “Materials and Methods”) described by Pinnix et al. (2001), using antibody 0443, which is highly sensitive in detecting AICD. We fractionated total brain homogenates into cytosol (Fig. 2 A), and membrane or nuclear fractions (Fig. 2 B); 20 μg protein was separated on a NuPAGE Bis-Tris 4–12% gel and probed with antibody 0443 that was raised against the COOH-terminal 20 residues of APP. As a positive control, we loaded cell extract from COS-1 cells that were cotransfected with AICD and Fe65 (lane 1). We readily detected AICD59 in the cytosolic fraction from FeCγ.25 mice (Fig. 2 A, top panel: lanes 6 and 7). A longer exposure of the same blot also revealed the presence of AICD in FeCγ.12 mice (bottom panel: lanes 4, 5), but not in the cytosolic fractions from Fe.27 (lanes 8 and 9) or nontransgenic control animals (lanes 2 and 3). The expression levels of AICD transgene parallel those of Fe65 transgene because FeCγ.12 mice express lower levels of both transgenes compared with FeCγ.25 animals (Fig. 1 C).


Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

Ryan KA, Pimplikar SW - J. Cell Biol. (2005)

AICD transgenic mice show barely detectable levels of AICD in the membrane fraction. (A) Cytosolic proteins from control (con) or two transgenic mice from indicated lines were separated on a 4–12% Bis-Tris NuPAGE gel, and the blots were probed with antibody 0443 after antigen retrieval as described in “Methods and materials.” Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). AICD band is clearly visible in FeCγ.25 mice (top panel, lanes 6 and 7), whereas a longer exposure shows the AICD protein present in FeCγ.12 mice (bottom panel, lanes 4 and 5), but not in control (lanes 2 and 3) or Fe.27 mice (lanes 8 and 9). (B) Brain homogenates from two control (con) transgenic mice from indicated lines were separated into cytosolic (Cyto.), membrane (Memb.), or nuclear (Nucl.) fractions, and the blots were probed with antibody 0443 as indicated above. Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). Although AICD was detected in the cytosol of only FeCγ transgenic mice (arrow), the membrane and nuclear fractions of all animals showed the AICD co-migrating band (arrows). (C) The AICD co-migrating band is absent in APP-KO mice. Brain membranes from two FeCγ.12 mice (lanes 2 and 3), APP-KO mice (lanes 4 and 5), and R1.40 transgenic mice expressing human APP with “Swedish mutation” (lanes 6 and 7) were probed with antibody 0443, as described above, on a NuPAGE gel. Top panel shows that the APP band is present in FeCγ.12 and in higher amounts in R1.40 mice, but is absent in APP-KO mice. Similarly, the middle panel shows the absence of APP-CTFs in the APP-KO mice. Note that the AICD co-migrating band (arrow) also is absent in APP-KO mice. (D) The AICD peptide becomes detectable only after antigen retrieval treatment (ART). Equal amounts of brain membrane proteins from two R1.40 mice were separated in duplicate on a NuPAGE gel. After electrophoretic transfer, the membrane was cut lengthwise to give two identical gels. One was exposed to boiling PBS for 5 min (ART), whereas the other was kept in PBS at room temperature (con). Arrow shows the AICD band, which is detectable only in the ART samples. (E) AICD band (arrow) is present in increased amounts in R1.40 transgenic mice compared with the controls (con) and is absent in APP-KO mice. Membrane fractions from indicated mice were probed with antibody 0443.
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fig2: AICD transgenic mice show barely detectable levels of AICD in the membrane fraction. (A) Cytosolic proteins from control (con) or two transgenic mice from indicated lines were separated on a 4–12% Bis-Tris NuPAGE gel, and the blots were probed with antibody 0443 after antigen retrieval as described in “Methods and materials.” Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). AICD band is clearly visible in FeCγ.25 mice (top panel, lanes 6 and 7), whereas a longer exposure shows the AICD protein present in FeCγ.12 mice (bottom panel, lanes 4 and 5), but not in control (lanes 2 and 3) or Fe.27 mice (lanes 8 and 9). (B) Brain homogenates from two control (con) transgenic mice from indicated lines were separated into cytosolic (Cyto.), membrane (Memb.), or nuclear (Nucl.) fractions, and the blots were probed with antibody 0443 as indicated above. Cell lysate from COS-1 cells cotransfected with AICD and Fe65 was run as a positive control (lane +). Although AICD was detected in the cytosol of only FeCγ transgenic mice (arrow), the membrane and nuclear fractions of all animals showed the AICD co-migrating band (arrows). (C) The AICD co-migrating band is absent in APP-KO mice. Brain membranes from two FeCγ.12 mice (lanes 2 and 3), APP-KO mice (lanes 4 and 5), and R1.40 transgenic mice expressing human APP with “Swedish mutation” (lanes 6 and 7) were probed with antibody 0443, as described above, on a NuPAGE gel. Top panel shows that the APP band is present in FeCγ.12 and in higher amounts in R1.40 mice, but is absent in APP-KO mice. Similarly, the middle panel shows the absence of APP-CTFs in the APP-KO mice. Note that the AICD co-migrating band (arrow) also is absent in APP-KO mice. (D) The AICD peptide becomes detectable only after antigen retrieval treatment (ART). Equal amounts of brain membrane proteins from two R1.40 mice were separated in duplicate on a NuPAGE gel. After electrophoretic transfer, the membrane was cut lengthwise to give two identical gels. One was exposed to boiling PBS for 5 min (ART), whereas the other was kept in PBS at room temperature (con). Arrow shows the AICD band, which is detectable only in the ART samples. (E) AICD band (arrow) is present in increased amounts in R1.40 transgenic mice compared with the controls (con) and is absent in APP-KO mice. Membrane fractions from indicated mice were probed with antibody 0443.
Mentions: We next determined the AICD levels by Western blot following a protocol (see “Materials and Methods”) described by Pinnix et al. (2001), using antibody 0443, which is highly sensitive in detecting AICD. We fractionated total brain homogenates into cytosol (Fig. 2 A), and membrane or nuclear fractions (Fig. 2 B); 20 μg protein was separated on a NuPAGE Bis-Tris 4–12% gel and probed with antibody 0443 that was raised against the COOH-terminal 20 residues of APP. As a positive control, we loaded cell extract from COS-1 cells that were cotransfected with AICD and Fe65 (lane 1). We readily detected AICD59 in the cytosolic fraction from FeCγ.25 mice (Fig. 2 A, top panel: lanes 6 and 7). A longer exposure of the same blot also revealed the presence of AICD in FeCγ.12 mice (bottom panel: lanes 4, 5), but not in the cytosolic fractions from Fe.27 (lanes 8 and 9) or nontransgenic control animals (lanes 2 and 3). The expression levels of AICD transgene parallel those of Fe65 transgene because FeCγ.12 mice express lower levels of both transgenes compared with FeCγ.25 animals (Fig. 1 C).

Bottom Line: APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm.In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined.Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

Show MeSH
Related in: MedlinePlus